Distinct daf-16 isoforms regulate specification of vulval precursor cells in Caenorhabditis elegans

FOXO transcription factors regulate development, longevity, and stress-resistance across species. The C. elegans FOXO ortholog, daf-16, has three major isoforms with distinct promoters and N-termini. Different combinations of isoforms regulate different processes. Adverse environments can induce dauer diapause after the second larval molt. During dauer, daf-16 blocks specification of vulval precursor cells, including EGFR/Ras-mediated 1˚ fate specification and LIN-12/Notch-mediated 2˚ fate specification. Using isoform-specific mutants, we find that daf-16a and daf-16f are functionally redundant for the block to the expression of 1˚ fate markers. In contrast, all three isoforms contribute to blocking the expression of 2˚ fate markers.

. daf-16 isoform-specific mutants express VPC specification markers during dauer (A) Vulval precursor cell (VPC) specification occurs in the L3 stage during continuous (non-dauer) development (Sternberg 2005). Six equipotential VPCs are named P3.p-P8.p. LIN-3/EGF is secreted from the anchor cell (AC) and a canonical EGFR/Ras/MAPK pathway specifies 1˚ vulval fate in P6.p (red). P6.p then expresses ligands for LIN-12/Notch, activating LIN-12 signaling in the adjacent cells, P5.p and P7.p. LIN-12 signaling specifies 2˚ vulval fate (blue). The remaining VPCs adopt a non-vulval fate (Sternberg 2005). (B) As larvae molt from L2d into dauer (L2d-D molt), 1˚ cell fate marker expression is observed in P6.p (red). However, this expression is lost within one day of entering dauer, such that larvae that have been in the dauer stage for one day ("1-day dauer") do not express 1˚ fate markers. 2˚ cell fate markers are not observed prior to dauer or during dauer in wild-type or daf-7 control larvae (Karp and Greenwald 2013). (C-D) The daf-7(e1372) allele was used to induce dauer formation and is present in all strains. Strains lacking one or more daf-16 isoform were scored for the presence or absence of expression of a 1˚ fate marker (arIs131 [lag-2p::yfp]) or 2˚ fate marker (arIs116 [lst-5p::yfp]). Individual dots represent the percent of larvae expressing the relevant marker in a single trial of 5-36 dauer larvae. The mean and standard deviation for each strain is shown. Letters above the data indicate statistical groupings, where strains that have a letter in common are not statistically different from each other. (Kruskal Wallis with Dunn's multiple comparison test, α = 0.05). Pvalues for all non-significant comparisons were > 0.99 with the following exceptions: In panel (C), daf-16(0) vs. daf-16(a), p = 0.08. In panel (D),f), vs. daf-16(f), p = 0.08. (C) Percent of 1-day dauer larvae that express the lag-2p::yfp 1˚ fate marker in f), mg54. Loss of daf-16a and daf-16f together recapitulates the phenotype of loss of all daf-16 isoforms in the null allele daf-16(0). (D) Percent of early dauer larvae that express the lst-5p::yfp 2˚ fate marker in f), mg54. Loss of each daf-16 isoform tested produces a phenotype intermediate between the control daf-7 strain and the daf-16(0) allele.

Description
Forkhead box O (FOXO) transcription factors regulate critical processes including development, metabolism, longevity, and stress-resistance. The C. elegans FOXO ortholog, daf-16, has been foundational for the discovery of many of these roles which are conserved across species (Kenyon et al. 1993;Murphy and Hu 2013;Tissenbaum 2018). daf-16 was discovered for its role in promoting entry into dauer diapause which occurs after the second larval molt in response to unfavorable environments (Cassada and Russell 1975;Vowels and Thomas 1992).
There are three major isoforms of daf-16: daf-16a, daf-16b, and daf-16d/f/h (hereafter called "daf-16f"), each with its own promoter (see Methods). These isoforms are expressed in distinct tissues and produce proteins that differ at the N-terminus (Lin et al. 1997;Ogg et al. 1997;Kwon et al. 2010). Of the three isoforms, daf-16b differs the most from the other two: while daf-16a and daf-16f are broadly expressed, daf-16b expression is found primarily in neurons, the pharynx, and somatic gonad. Furthermore, DAF-16a and DAF-16f have more amino acids in common, including the entire DNA-binding domain, whereas a portion of the DAF-16b DNA-binding domain comes from a distinct exon (Murphy and Hu 2013).
The molecular differences between isoforms appear to correlate with their biological functions. daf-16a and daf-16f together regulate longevity, dauer formation, and stress-resistance, with little to no input from daf-16b (Lee et al. 2001;Kwon et al. 2010;Chen et al. 2015). In contrast, daf-16b regulates neuronal morphology and behavior (Christensen et al. 2011;Sun et al. 2019). However, there are some examples of shared roles between daf-16b and other isoforms. daf-16a and daf-16b both contribute to innate immunity in adults, though daf-16a plays the predominant role (McHugh et al. 2020). Finally, all three isoforms contribute to preventing expression of an adult cell fate marker in the hypodermis of dauer larvae (Wirick et al. 2021).
During dauer, daf-16 is part of a mechanism to block specification of vulval precursor cells (VPCs) (Karp and Greenwald 2013). Six VPCs, P3.p-P8.p, are born in the L1 stage, remain multipotent in the L2 stage, and are specified to one of two vulval fates in the L3 stage. LIN-3/EGF secreted from the anchor cell specifies 1˚ fate in the underlying VPC, P6.p. P6.p then signals to its neighbors via LIN-12/Notch to specify 2˚ cell fate. VPCs that do not receive either signal take on a non-vulval fate (3˚) (Fig. 1A) (Sternberg 2005). In the dauer life history, 1˚ fate specification begins prior to dauer formation, but this specification is erased within one day and thereafter dauer larvae do not express either 1˚ or 2˚ cell fate markers (Fig. 1B) (Karp and Greenwald 2013). The block to 1˚ fate occurs at or upstream of Ras activation, whereas the block to 2˚ fate appears to occur downstream or in parallel to activation of LIN-12/Notch (Karp and Greenwald 2013;O'Keeffe and Greenwald 2022). However, dauer larvae with a null allele of daf-16 (daf-16(0)) express both 1˚ and 2˚ markers during dauer, indicating that daf-16 opposes both 1˚ and 2˚ cell fate specification (Karp and Greenwald 2013).
To determine which daf-16 isoforms are responsible for blocking VPC specification during dauer, we took advantage of existing isoform-specific mutants. These mutations eliminate either daf-16a, daf-16f, or both daf-16a and daf-16f (daf-16a,f), without affecting the expression of the remaining isoforms (Chen et al. 2015). Specifically, the daf-16f allele, tm6659, deletes the exons specific to the daf-16f isoform. Two similar alleles, tm5030 and tm5032, denoted here as daf-16(a1) and daf-16(a2), each delete a portion of the daf-16a-specific exon and cause frameshift mutations. Finally, the daf-16(a,f) allele, mg54, is a nonsense mutation in an exon that is common to the daf-16f and daf-16a isoforms but upstream of the first daf-16b exon (Chen et al. 2015). We crossed these alleles into strains containing either 1˚ or 2˚ cell fate markers. All strains also contained the daf-7(e1372) mutation to induce dauer formation in daf-16 mutants (Vowels and Thomas 1992;Larsen et al. 1995;Karp 2018).
To determine which daf-16 isoforms are involved in blocking 1˚ fate specification, we used lag-2p::yfp as our 1˚ fate marker. lag-2 encodes a LIN-12 ligand expressed by P6.p after commitment to 1˚ fate (Chen and Greenwald 2004;Zhang and Greenwald 2011). Consistent with prior work, in control daf-7 dauer larvae, lag-2p::yfp was expressed as larvae molt into dauer, but expression faded over the first day in dauer as multipotency was re-established. In contrast, daf-16(0); daf-7 larvae continued to express lag-2p::yfp at high penetrance (Fig. 1C) (Karp and Greenwald 2013). While loss of daf-16a caused only minimal lag-2p::yfp expression in 1-day dauer larvae and loss of daf-16f was not different from the control daf-7 strain, loss of both isoforms together produced a phenotype statistically indistinguishable from the null allele (Fig. 1C). With the caveat that we were not able to directly test a daf-16(b) allele, these data suggest that isoforms a and f function together to block 1s pecification and that daf-16b plays at most a minor role in this process. This role is similar to the requirement for those isoforms to regulate dauer formation, lifespan, and stress resistance (Lee et al. 2001;Kwon et al. 2010;Chen et al. 2015).
To determine which daf-16 isoforms are involved in blocking 2˚ fate specification, we used lst-5::yfp as our 2˚ marker. lst-5 is a direct transcriptional target of LIN-12 and is expressed in P5.p and P7.p after commitment to 2˚ fate (Li and Greenwald 2010;Underwood et al. 2017). Control daf-7 larvae that had recently molted into dauer expressed very little lst-5p::yfp in P5p or P7.p. In contrast, daf-16(0); daf-7 larvae displayed penetrant lst-5p::yfp expression at this time ( Fig. 1D) (Karp and Greenwald 2013). Loss of daf-16a, daf-16f, or both daf-16a and daf-16f produced an intermediate phenotype such that the penetrance of lst-5p::yfp expression was not statistically different from either the daf-7 or the daf-16(0); daf-7 controls (Fig.  1D). These data suggest that all three isoforms contribute to blocking 2˚ cell fate specification. This role is similar to the requirement for all three isoforms to block expression of the col-19p::gfp adult cell fate marker in the lateral hypodermis of dauer larvae (Wirick et al. 2021).
Taken together, we have found distinct requirements for daf-16 isoforms to block 1˚ and 2˚ cell fate specification during dauer. Promoter swap experiments indicate that differences in expression explain requirements for different isoforms for the regulation of lifespan and dauer formation (Lee et al. 2001;Kwon et al. 2010). daf-16 is required in VPCs to block expression of the 1˚ fate marker used in this study; the focus of daf-16 activity for its role in 2˚ fate is unknown (Karp and Greenwald 2013). Furthermore, in wild-type larvae, lag-2p::yfp is expressed during the L2d-dauer molt and downregulated during dauer whereas lst-5p::yfp is not expressed in the L2d-dauer molt or dauer (Karp and Greenwald 2013). This difference in expression raises the possibility that the block to 1˚ fate specification occurs later than the block to 2˚ specification. It will be interesting to investigate whether the distinct requirements identified in this study are due to differences in expression of daf-16 isoforms, either spatially or temporally, differences in the focus of daf-16 activity for 1˚ and 2˚ fate, or the unique N-terminus of each daf-16 isoform.

Strains and maintenance
C. elegans strains were maintained at 20˚C on NGM plates with E. coli strain OP50 as a food source.

Dauer induction
The daf-7(e1372) allele was present in all strains and used to induce dauer formation (Vowels and Thomas 1992;Larsen et al. 1995;Karp 2018). Ten to thirty gravid adult hermaphrodites of the desired strains were added to seeded plates at 24˚C or 25˚C. The adults were allowed to lay eggs for 5 hours at 25˚C. The adults were removed, and the embryos were incubated at 25˚C for a further 48 hours for "early dauers" or 72 hours for "1-day dauers". At 25˚C, daf-7 larvae molt into dauer at approximately 48 hours after egg-laying (Karp 2018). To ensure larvae were within the dauer stage, prior to scoring, putative dauer larvae were added to an NGM plate seeded with 10x concentrated OP50 mixed with red fluorescent beads (Sigma L3280) at a 1:1000 ratio (v/v) of beads to 10x OP50. Larvae were incubated on bead-containing plates for 30 minutes. Dauer larvae, including daf-16(0) dauer larvae, do not ingest food and thus do not take up any fluorescent beads during this time (Nika et al. 2016); therefore, larvae that lacked beads were used for experiments. To ensure larvae were not still within the L2d-dauer molt, DIC optics were used to confirm radial constriction and fully formed dauer alae.

Microscopy
Larvae were anesthetized in 0.1M levamisole on 2% agarose pads. Worms were imaged using a Zeiss AxioImager D2 compound microscope with a 63x objective, an AxioCam MRm Rev 3 camera, and Zen software (Zeiss). To avoid bleaching, DIC optics were used to locate the relevant VPCs followed by snapping a single image of DIC and YFP channels using a 500ms exposure time for YFP. Images were categorized as on or off, where on indicated detectable YFP expression in the relevant VPCs in the image. Controls were performed in parallel with isoform-specific mutants, and each strain was scored by 2-3 independent researchers. The combined data from all researchers are shown in Figure 1.

daf-16 isoforms
The daf-16 isoforms described in this study are those that have been defined and characterized in the literature (Lin et al. 1997;Ogg et al. 1997;Lee et al. 2001;Kwon et al. 2010;Chen et al. 2015). All of these isoforms contain a Forkhead DNA binding domain, phosphorylation sites for AKT, and N-terminal phosphorylation sites. However, the sequences at the N-terminus vary between isoforms (reviewed in Murphy and Hu 2013 (Chen et al. 2015). However, the daf-16(a,f) mutation, mg54, is an early stop codon that should disrupt isoforms R13H8.1l.1 and R13H8.1m.1 (Ogg et al. 1997